| 摘要: |
| [目的]克隆和表达奶牛乳腺炎金黄色葡萄球菌纤连蛋白连接蛋白A(Fnbp A)的功能基因.[方法]采集奶牛急性乳腺炎乳样,分离金黄色葡萄球菌,提取其DNA作为模板,利用设计合成的特异性引物,对金黄色葡萄球菌进行Fnbp A功能基因的PCR扩增.回收目的基因并连接到T载体,鉴定后进行测序,然后将Fnbp A基因连接到pET-32a(+)质粒中,经鉴定后转化大肠杆菌BL21感受态细胞,IPTG诱导后采用SDS-PAGE分析蛋白质的表达情况.[结果]PCR产物经电泳成像,仅金黄色葡萄球菌在1.7 kb处出现特异性条带,测序发现其与GenBank公布的FnbpA序列的同源性为98%;蛋白诱导表达SDS-PAGE分析发现,在80 ku处出现特异性条带.[结论]试验成功地克隆到Fnbp A基因,并在大肠杆菌中获得高效表达,蛋白量为33.4%,为进一步研究金黄色葡萄球菌粘附素基因工程疫苗奠定了基础. |
| 关键词: 金黄色葡萄球菌 乳腺炎 粘附素 纤连蛋白连接蛋白A |
| DOI： |
| 分类号:S827.2 Q78 |
| 基金项目:教育部高等学校科技创新工程重大项目（706039）；山东省“三0”工程项目 （2003-3009）；山东省农科院高技术自主创新基金项目（2006YCX027）;山东省农科院重大成果培育基金项目（2006YCG012） |
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| Cloning and expression of the functional Fnbp A gene of Staphylococcus aureus adhesin |
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LI Jun-ying CHEN Hong LAN Xian-yong MIN Ling-jiang LEI Chu-zhao HU Shen-rong
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| Abstract: |
| 【Objective】 To clone and express the gene of fibronectin-binding protein (Fnbp A),the Staphylococcus aureu were isolated from the milk samples collected from dairy cows with peracute clinic mastitis.【Method】 The DNA of the Staphylococcus aureus was extracted with classical technique to be template.The Fnbp A gene was amplified by PCR and cloned into plasmid of pMD-18-T.The recombinant plasmid was transferred into competent E.coli.After the recombinants were screened and identified by restrition analysis,the cloned gene was sequenced.The Fnbp A gene was cloned into pET-32a(+),with the inducement of IPTG,it is expressed at high levels in E.coli.BL21.【Result】 Result showed that a specific protein band was found in SDS PAGE with the molecular weight of approximately 80 ku,which accorded with the anticipated result. 【Conclusion】 The turnout of the protein was about 33.4% of the total protein of the bacterium analyzed with the BandScan 5.0.The successful expression of Fnbp A in E.coli BL21 constituted a solid foundation for Staphylococcal vaccine development. |
| Key words: Staphylococcus aureus mastitis adhesin Fibronectin-binding protein A |
